Assessment of risk of aneuploidy

ABSTRACT

The present disclosure relates generally to methods and materials for use in detecting abnormalities of the number of whole chromosomes or chromosome regions (aneuploidy). It has particular utility for assessing the risk of aneuploidy of eggs (i.e., oocytes), fertilised eggs or embryos developed therefrom in the context of in vitro fertilisation.

TECHNICAL FIELD

The present disclosure relates generally to methods and materials foruse in detecting abnormalities of the number of whole chromosomes orchromosome regions (aneuploidy). It has particular utility for assessingthe risk of aneuploidy of eggs (i.e., oocytes), fertilised eggs orembryos developed therefrom in the context of in vitro fertilisation.

BACKGROUND ART

In normal female meiosis the precursor cells of the ova multiply andthen reduce the number of chromosomes to one half set in each gamete intwo specialised meiotic divisions.

Meiosis is initiated in the fetal ovary before birth during the earlydevelopment of the female germ cells (primary oocytes), which willeventually form mature eggs (or oocytes, the terms are usedinterchangeably) in the adult female.

To reduce the number of chromosomes from the normal (euploid) 23 pairsof homologouschromosomes (one of each pair inherited from the father andone from the mother, so 46 in total) to 23 single chromosomes, there isone round of DNA replication in which each chromosome is duplicated intotwo sister chromatids followed by two specialised meiotic divisions,meiosis I and II.

Following replication, the two homologous chromosomes of each pair ‘pairup’ and a single bivalent chromosome forms in which all four sisterchromatids are tightly bound together. This allows a limited number ofbreaks in the DNA strands of adjacent non-sister chromatids to‘crossover’ and re-join the other chromatid, leading to non-recombinant(no exchange) and recombinant chromatids and generating geneticvariation.

As the cell divides at the end of meiosis I, one homologous chromosomeof each pair is pulled into the first polar body and the other into thesecondary oocyte, which therefore now has 23 chromosomes each with twosister chromatids.

In meiosis II, following fertilisation of the oocyte by a sperm cellcontaining the paternal half set of chromosomes, the two sisterchromatids of each chromosome finally separate and segregate into thesecond polar body and the fertilised oocyte (now a zygote). The zygotetherefore inherits 23 single maternal chromatids (now ‘chromosomes’).

Aneuploidy is defined as an abnormal number of whole chromosomes orparts of chromosomes causing a genetic imbalance. The most frequent andclinically significant aneuploidies involve single chromosomes (strictly‘aneusomy’) in which there are either three (‘trisomy’) or only one(‘monosomy’) instead of the normal pair of chromosomes per somatic cell.

Chromosome aneuploidy is a major cause of pregnancy loss and abnormalpregnancy with live births and increases exponentially with maternal agein the decade preceding menopause (Hassold and Hunt, 2001). Mostautosomal aneuploidies and all autosomal monosomies are lethal, only asmall number of trisomies are compatible with full term developmentoften with severe congenital abnormalities.

A similar pattern of aneuploidy occurs in pregnancies following assistedconception using in vitro fertilisation (IVF) (Spandorfer et al., 2004).Furthermore, microarray based comparative genomic hybridisation (e.g.,array CGH) analysis has shown that a majority of human oocytes in womenof advanced maternal age (average age 40) are aneuploid, most withmultiple aneuploidies (Handyside et al., 2012).

Currently, human oocytes can be tested for aneuploidy using whole genomeamplification (WGA) of both the first and second polar bodies (PB1 andPB2, respectively) by microarray based comparative genomic hybridisation(array CGH). Array CGH is a methodology, which compares the amount ofDNA hybridising to DNA probes spaced typically at 1 Mb intervals acrossthe genome, i.e. across each chromosome, in test and control DNAlabelled with green and red fluorochromes (24Sure™, BlueGnome Ltd;www.24Suretest.com), for example. With human oocytes, WGA products fromthe two polar bodies are labelled and hybridised and the signalintensities compared to control male and female DNA labelled in oppositefluorochromes.

The polar bodies are by-products of the two meiotic divisions, meiosis Iand II, and since they do not form part of the embryo they can beremoved with minimal effect using published methods well known to thoseskilled in the art. However, biopsying both polar bodies from eachoocyte is labour intensive for clinics and multiple arrays are requiredto test each oocyte.

Other methods for detecting aneuploidy have been proposed as describedherein.

For example, the presence or absence of each chromosome in polar bodiescan be detected by multiplex PCR of panels of chromosome specificsequences (Advalytix, Beckman Coulter; www.advalytix.com/advalytix/).However amplification bias makes it difficult to accurately quantify theproducts, thereby limiting possible application for aneuploidy testingin polar bodies.

Limiting dilution into separate wells and digital PCR can be used tocount the number of chromatids. However by virtue of the steps involved,this methodology can be technically challenging, and has not yet beenextensively validated (for example, see publication WO2011/138750 of theMRC et al).

Thus it can be seen that novel, less complex, methods for assessing therisk of aneuploidy of eggs, fertilised eggs or embryos developedtherefrom would provide a contribution to the art.

DISCLOSURE OF THE INVENTION

This disclosure provides a novel approach to assessing whether eggs (andthus fertilised eggs or embryos developed therefrom) are at increasedrisk of aneuploidy of maternal meiotic origin, by analysing the firstpolar body (PB1), optionally with other measures as described below.

Centromeric Heterozygosity (CH) in PB1 for Use in Assessing Risk

More specifically, it has been determined that an increased risk oferrors in meiosis II, which would lead to aneuploidy in the fertilisedegg, can be assessed by measuring total heterozygosity or centromericheterozygosity (CH) in PB1 only, optionally with other measures asdescribed below. In particular, where CH is present or elevated, andeven where the secondary oocyte appears euploid (having 2n sisterchromatids) there is an increased risk of aneuploidy followingsegregation in meiosis II, compared to where CH is not detected, or isat a low level.

As explained herein, the normal situation for PB1 is that it contains achromosome consisting of predominantly or entirely homozygous sisterchromatids replicated from one of the homologous chromosomes from themother. In such a situation CH would be very low or absent.

Using CH this normal situation can be distinguished in PB1 from theabnormal presence of one or more chromosomes which comprise two or morenon-sister chromatids, the chromatids being derived from both of thehomologous maternal chromosomes. Such non-sister chromatids can beheterozygous at various loci, and importantly may display at least somelevel of CH.

Because significant CH would not arise merely from recombination, anovel system has been developed in which its presence can be used toinfer a risk of aneuploidies of various types, being manifest aftermeiosis II.

The novel systems described herein may be employed to estimate ploidy(euploid, aneuploidy) status of the egg (and fertilised egg, andembryo).

It will be appreciated from the disclosure herein that embodiments ofthe disclosure are directed at assessing risk. Thus a proportion ofchromosomes in which CH is present or elevated in PB1 may not result inaneuploidies, because the pattern of segregation in meiosis IInevertheless results in a euploid egg. However, the inventionnevertheless has significant utility in practice because it is knownthat many oocytes (and resulting fertilised eggs and embryos) will havemultiple aneuploidies. The present disclosure provides a clinicallyuseful methodology for grading or selecting eggs or embryos before orafter fertilisation based on relative risk.

In particular, where there is a panel of PB1s being assessedcorresponding to a panel of oocytes, the disclosure herein provides amethodology based on CH for selecting one or more oocytes in which thelikelihood of errors having occurred during meiosis I is reducedcompared to the other oocytes. This is discussed in more detail below.

Number of Crossovers in PB1 for Use in Assessing Risk

Furthermore, parallel analysis of oocytes and PB1 by the presentinventors has revealed that the total number of crossovers in the firstpolar body (of all the chromosomes assessed) can be correlated with ageneral tendency to aneuploidy.

More specifically total crossovers in some or preferably all of thechromosomes in PB1 can be estimated from the number and position ofblocks of heterozygosity, and this can be used as a proxy for the totalnumber of recombination events (crossovers, meiotic exchanges; the termsare used synonymously unless context demands otherwise) in therespective oocyte.

It is known in the art that a low frequency of recombination isassociated with abnormalities in chromosome segregation and, inparticular, increased levels of non-disjunction, leading to aneuploidy(see Hassold, 2001). Therefore a relatively low number of crossovers inthe first polar body can be indicative of tendency to aneuploidy of theoocyte.

For example, the detection of less than or equal to 50 recombinationevents across all chromosomes in PB1 may be taken to indicate asignificantly higher risk in the corresponding oocyte than whereaverage, or above average numbers (>75) are detected. The detection ofequal to or between 51 and 74 events across all chromosomes in PB1 maybe taken to indicate an above average risk. The detection of greaterthan or equal to 75 would be deemed average risk (i.e. no adverseinference to be drawn, based purely on total number of recombinationevents).

Location of Crossovers in PB1 for Use in Assessing Risk

It is known that chromosomes with a single proximally (close tocentromere) or distally located recombination are more likely tonon-disjoin than were those with more medially positioned recombinations(see Hassold, 2001). Accordingly where such a single recombination eventis detected in PB1, this may also suggest a general tendency toaneuploidy in relation to that chromosome.

Other Utilities of High Resolution SNP Mapping of PB1

As shown in the Examples below, high resolution SNP mapping of PB1 canreveal de novo structural chromosome abnormalities. In particular, theinventors have shown that chromosomal structural defects in the oocytewere reflected in PB1.

Therefore in addition to CH, analysis of the number of crossovers insome or all of the chromosomes of PB1, as well as the location ofcrossovers, provides an alternative, or additional, methodology forassessing risks of aneuploidy. As described herein, that has utility(inter alia) in oocyte or embryo selection and/or as a diagnostic orprognostic indicator of the likelihood of successful pregnancy.

PREFERRED EMBODIMENTS

In preferred embodiments a method of assessing heterozygosity or CH iscarried out by whole genome amplification (WGA) and genotyping forheterozygous loci (e.g., such as single nucleotide polymorphisms, SNPs),which for CH must be close to and flanking the centromeres of eachchromosome in PB1.

Where whole genome amplification and SNP genotype analysis is performed,it may also be desirable to phase the SNPs of the maternal chromosomes.This permits the existence of heterozygosity around the centromere inPB1 to be inferred even in the event of random allele dropout at theheterozygous maternal loci, again distinguishing the ‘normal situation’from the abnormal presence of two or more non-sister chromatids. Asnoted above, it will generally be preferred to perform SNP genotype orhaplotype analysis across the entire genome, in order to assess thenumber and\or location of recombination sites.

Whole genome amplification and genotype analysis can additionally,optionally, be used to determine chromosomal aneuploidy resulting frommeiosis I errors—for example the absence of any centromeric DNA for agiven chromosome would also identify a risk of aneuploidy.

The method differs from prior art quantitative methods, such as arrayCGH to assess copy number change in PB1, since those methods would onlyidentify errors in meiosis I (resulting in aneuploidy in the secondaryoocyte). In the present invention potential errors and risks arisingfrom meiosis I and II can be identified by PB1 analysis alone, reducingthe time and cost. Furthermore, genotyping is in principle more reliablethan quantitation because it is unaffected by amplification biasfollowing WGA.

Some aspects and embodiments of the present invention will now bediscussed in more detail:

In one aspect there is provided a method of investigating the ploidystatus of a human egg (or assessing the risk of chromosomal aneuploidyof maternal meiotic origin in the egg), the method comprising assessingthe heterozygosity for one or more (preferably all) the chromosomes ofthe first polar body of the human egg.

The data not only therefore provides information about centromericheterozygosity (CH), but also total crossovers, and location ofcrossovers. These can in turn be used to infer a higher likelihood ofaneuploidy—which can, for example, be inferred by a relatively low totalnumber of cross-over events, or signature single recombination eventsnear the centromere or telomere of a given chromosome.

In a preferred aspect there is provided a method of investigating theploidy status of a human egg (or assessing the risk of chromosomalaneuploidy of maternal meiotic origin in the egg), the method comprisingassessing the presence or degree of centromeric heterozygosity (CH) forone or more chromosomes of the first polar body of the human egg.

As explained herein, assessing the polar body can predict the risk ofaneuploidy in the corresponding egg, and thus also the fertilised egg orembryo following meiosis II.

Preferably a plurality or panel or chromosomes is assessed.

Preferably a plurality of different first polar bodies is assessed, andthe corresponding eggs, or fertilised eggs or embryos developedtherefrom, are graded according to risk as described herein. This may inturn be used as part of a method of IVF.

In particular the presence of a higher level of centromericheterozygosity (CH) in a first polar body can be used to infer a higherrisk of said chromosomal aneuploidy in the corresponding egg or embryodeveloped therefrom compared to an absence or lower level of CH in afirst polar body.

The assessment of risk by CH can also, additionally, utilise:

(i) total crossovers in PB1;(ii) location of crossovers of chromosomes in PB1.

In one embodiment, methods disclosed herein are based on using CH todistinguish a ‘normal’ PB1 which contains a chromosome consisting ofhomozygous sister chromatids replicated from one of the homologouschromosomes from the mother, from an ‘abnormal’ PB1 wherein thechromosomes comprise two or more non-sister chromatids, collectivelybeing derived from both of the homologous maternal chromosomes. Thisinformation, optionally with crossover number and\or location analysis,may be used to infer a risk of aneuploidies, of various types, beingmanifest after meiosis II.

The PB1 may be from a post-pubescent human female of any age. Optionallythe PB1 may be from a human female of 35 years or older. Practically,the PB1 may be from a human subject (of any age) who may have, or issuspected of having, fertility problems or has or carries an inheritabledisease. The PB1 may be from a human subject undergoing IVF treatment.

In one aspect, the present invention provides a method for in vitrofertilisation utilising a fertilised egg or embryo predicted to be atrelatively low risk of aneuploidy according to results derived bypracticing a method of the invention. “Relatively low risk” in thiscontext means by comparison with other fertilised eggs or embryos forwhich the corresponding PB1s have also been assessed according to amethod of the invention.

Unless context demands otherwise, where “a” or “the” chromosome isreferred to herein in respect of SNP genotyping, this refers to typing aplurality of copies of that chromosome (or corresponding chromatid orchromatids) which are present in the target cell.

In one embodiment at least 2, 3, 4, 5 or 6 or all of the humanchromosomes selected from the following group are assessed: X, 22, 21,18, 16 and 13. Imbalances in any of these chromosomes may be associatedwith viable but abnormal pregnancies. A preferred combination ofchromosomes for assessment comprises chromosomes 21, 18 and 13.

Preferably a total of at least 5, 10, 15 or 20 chromosomes are assessed.

In one embodiment the entire genome of PB1 is assessed (i.e. the CH ofall 23 chromosomes is analysed). This is particularly desired wheretotal crossover number in PB1 is to be assessed, in order to detect theexistence of a low frequency of crossovers or recombination.

Preferably total heterozygosity and\or CH is assessed in the methodsdescribed herein by genotyping a plurality of SNP loci. These includegenome sequencing e.g. by methods such as so called “Next Generationsequencing” (NGS)—see e.g. www.illumina.com.

Preferably total heterozygosity and\or CH assessment is preceded by WGA.

The assessment based on total heterozygosity and\or CH may or may not beused in conjunction with (prior to, or subsequently to) quantitationmethods.

As discussed herein, SNPs can be interrogated using conventionaltechniques. This may be preceded by one or more conventionalamplification steps.

In one embodiment the invention first comprises identifying the positionof centromeres in the sequence of consecutive SNPs across eachchromosome to be assessed.

The invention may thus comprise interrogating closely adjacent biallelicSNPs flanking the centromeres of the chromosomes of PB1.

In one embodiment equal to or at least 25, 30, 40, 50, 75, 100, 200,300, 400, 500 or more SNPs flanking the centromere are interrogated.Preferably at least around 200 SNPs are assessed.

This number may be interrogated on each of the p and q arms of eachchromosome (or q arms only for acrocentric chromosomes).

However for individual chromosomes a lesser number may besufficient—this can be assessed by those skilled in the art according tothe preferred method of typing and the accuracy associated with it andwith any optional method of amplification employed.

In one embodiment 200 SNPs across a 2 Mb region of the chromosome within5-10 Mb of the centromere are analysed.

Once the SNPs have been interrogated, the proportion of heterozygous(‘AB’) SNPs can be established.

A proportion of these interrogated SNPs within the region genotypedflanking the centromere exceeding e.g. 50, 60, 70, 80, 90, or 100% canbe taken as being highly heterozygous (“high CH”) in accordance with themethods described herein.

Recombination is significantly less frequent near the centromere (seeLynn, A., Ashley, T., and Hassold, T. (2004). Variation in human meioticrecombination, Annu Rev Genomics Hum Genet 5, 317-49). Thus it can beinferred that high levels of CH are associated with the presence ofhomologous maternal chromosomes as opposed to sister chromatids. In thisrespect it is highly preferable to use maternal genomic DNA as areference when performing the methods or other aspects of the invention.

Thus it is preferable to calculate the proportion of maternalheterozygous (AB) SNPs which are also heterozygous in the first polarbody. In this case equal to, or at least, 5, 10, 15, 20, 25, 50, 100,200 or more heterozygous (in the maternal cell) SNPs per flank may beassessed.

A number of these e.g. exceeding 10, 20, 30, 40, or a proportion ofthese e.g. exceeding 50, 60, 70, 80, or 90% can be taken as being“highly” heterozygous in accordance with the methods described herein.It will be understood that even a minimal block (of even 5 or more, or10 or more) of heterozygous SNPs within the centromeric region genotyped(proximal) can be indicative of high heterozygosity in accordance withthe methods described herein. The presence of such heterozygous SNPsfound on both sides of the centromere would provide additional, but notrequired, confirmation. Those skilled in the art will appreciate fromthe disclosure herein that the chosen threshold will be that whichaccommodates the small likelihood of recombination occurring close tothe centromere in the region genotyped and\or any ‘miscalls’ or ‘alleledrop-ins’, the likelihood of which will depend on the detectiontechnology adopted. The present inventors have determined thatbackground drop-ins (i.e. where a homozygous allele may be called asheterozygous) can be kept to extremely low levels, being less than 5% inthe examples tested. Thus a threshold of great than 5% heterozygosity ispreferred.

As explained in more detail herein, by phasing SNPs on the maternalchromosomes, any effect of random allele dropout at heterozygousmaternal loci can be minimised. Thus in one embodiment the presence or ahigher level of CH in a first polar body, for example as compared to athreshold proportion of heterozygous alleles in a normal first polarbody, and can be correlated with an increased risk of chromosomalaneuploidy in the corresponding egg or embryo resulting from, forexample, mis-segregation of single chromatids in meiosis II. Asexplained in the Examples this may follow bi-orientation of bivalentchromosomes in metaphase I of meiosis I. Said mis-segregation can leadto an egg lacking the relevant chromosome, or in which both homologousmaternal chromosomes are present (see e.g. FIG. 4).

In another embodiment the presence or a higher level of CH in a firstpolar body may be correlated with an increased risk of chromosomalaneuploidy in the corresponding egg or embryo resulting fromnon-disjunction of bivalent chromosomes in meiosis I leading to an eggin which both homologous maternal chromosomes are present (see e.g. FIG.2)

In a different embodiment a lack of centromeric alleles in a first polarbody implies a higher risk of chromosomal aneuploidy in thecorresponding egg or embryo developed therefrom (compared to thepresence of centromeric alleles). Said absence may imply non-disjunctionof bivalent chromosomes in meiosis I which leads to an egg lacking therelevant maternal chromosome (see e.g. FIG. 2).

In certain embodiments it may be desired to additionally subject thenucleic acid in PB1 to quantitation e.g. using quantitative fluorescentPCR or any other known method. This can provide, for example, moredetailed information regarding the ploidy of the fertilised egg orembryo.

For example where the presence or a higher level of CH has beendetermined, and the first polar body is determined as being euploidhaving 2n chromatids per chromosome, the presence or a higher level ofCH can imply an increased likelihood of chromosomal aneuploidy ofmaternal meiotic origin resulting from the 2nd meiotic division of thesecondary oocyte following fertilisation (see e.g. FIG. 4).

Where an absence or lower level of CH has been determined, and the firstpolar body is determined as being aneuploid having 0n chromatids for oneor more chromosomes, this can imply chromosomal aneuploidy of maternalmeiotic origin resulting from the 1st meiotic division of the primaryoocyte e.g. resulting from non-disjunction of bivalent chromosomes (seee.g. FIG. 2).

Where the presence or a higher level of CH has been determined, and thefirst polar body is determined as being aneuploid having 4n chromatidsfor one or more chromosomes, this can imply chromosomal aneuploidy ofmaternal meiotic origin resulting from the 1st meiotic division of theprimary oocyte e.g. resulting from non-disjunction of bivalentchromosomes (see e.g. FIG. 2).

Where the presence or a higher level of CH has been determined, and thefirst polar body is determined as being aneuploid having 3n chromatidsfor one or more chromosomes, this can imply chromosomal aneuploidy ofmaternal meiotic origin resulting from the 1st meiotic division of theprimary oocyte e.g. resulting from premature predivision of chromatids(see e.g. FIG. 3)

Where an absence or lower level of CH has been determined, and the firstpolar body is determined as being aneuploid having 1 n chromatids forone or more chromosomes, this can imply chromosomal aneuploidy ofmaternal meiotic origin resulting from the 1st meiotic division of theprimary oocyte e.g. resulting from premature predivision of chromatids(see e.g. FIG. 3).

This is summarised in Table 1 below. Referring to the Table, the normalfirst polar body centromeric SNP genotype would be AA or BB (no CH) andthe normal maternal chromosome copy number in a zygote is 1.

Any aspect or embodiment of the invention may further embracecharacterising a zygote (fertilised egg, or embryo) as having either anormal segregation pattern or one of the abnormal segregation patternsas described in Table 1 by assessing the PB1 CH and optionallychromosome copy number e.g. in accordance with the combinations shown inTable 1.

Any aspect or embodiment of the invention may further embrace predictingthe maternal chromosome copy number in a zygote (fertilised egg, orembryo) as being any of:

0120 or 11 or 20, 1 or 2by assessing the PB1 CH and preferably chromosome copy number e.g. inaccordance with the combinations shown in Table 1.

TABLE 1 First polar First polar Maternal body body chromosomeSegregation FIG. centromeric chromosome copy number pattern referenceSNP copy number in zygote Normal 2.1 AA or BB 2 1 Non- 2.2 AA or BB 2 2disjunction − 2.3 AA or BB 2 0 Meiosis II Non- 2.4 No result 0 2disjunction − 2.5 AB * 4 0 Meiosis I Premature 3.1 AB * 3 0 predivision− 3.2 AB 3 1 Meiosis I 3.3 AA or BB 1 1 3.4 AA or BB 1 2 Biorientation4.1 AB 2 1 of 4.2 AB * 2 0 chromosome − 4.3 AB * 2 2 Meiosis III

Utility and Prognostic Applications

A non-limiting list of utilities for the present invention includes PB1analysis alone as a screen to prioritise embryos for transfer andprovide prognostic information to the prospective parent, and PB1followed by PB2 for aneuploidy diagnosis and embryo selection, whereincreased accuracy is required.

Currently, the selection of one or more embryos, for example fortransfer in IVF treatments, may be carried out on the basis of arelatively crude morphological scoring system which categorises eachembryo, typically on a scale of 1-4. The score is based on the number ofcells an embryo should have attained by the time of the observation, howevenly the cells of the embryo have divided, the presence of any visiblenuclear abnormalities, and so on. The relevant number of embryos is thenselected with either the best scores or co-equal scores.

The use of total heterozygosity and\or CH screening provides animportant addition or alternative to these morphological ormorphokinetic methods. As explained above, the detection of totalheterozygosity and\or CH can highlight the increased possibility ofaneuploidy outcomes (see e.g. scenarios 2.5, 3.1, 4.2 and\or 4.3 inTable 1, which are marked with an *). This information may thus be usedto estimate a risk of aneuploidy and\or simply to select or grade eggsaccording to relative risk. The total heterozygosity and\or CH analysismay be done in combination with chromosome copy number. However, as isapparent from 4.2 and 4.3, this assessment can have utility even where aquantitative assessment has shown a normal copy number for the relevantPB1 or when morphological or morphokinetic analysis does not permiteffective selection.

Preferably in conjunction with known relationships or correlationsbetween populations of women of different ages and types of infertility,and optionally the other assessments described herein, the incidence oftotally heterozygous and\or CH chromosomes in PB1s of their oocytes isused as a general prognostic indicator of increased risk of aneuploidyand therefore reduced chance of pregnancy. Based on the above analysis,an actual risk of aneuploidy can be estimated by inferring thelikelihood of meiotic errors of the types shown in the Figures and\or byusing experimental evidence in which totally heterozygous and\or CHchromosomes are followed up by copy number analysis by, for example,array CGH in the corresponding embryos, to establish actual incidence ofabnormal segregation in meiosis.

More specifically, it has been demonstrated that most errors (>90%) inoocytes are caused by premature predivision of sister chromatids (FIG.3) and very few are caused by non-disjunction of whole chromosomes (FIG.2). If CH is identified for a particular chromosome it is thereforehighly likely to be caused by premature predivision or biorientation(see Table 1 above). In the former case, a single chromatid is presentin the secondary oocyte and will segregate at random to either PB2 orthe fertilised oocyte on completion of the 2nd meiotic divisionfollowing fertilisation. From first principles, the probability ofaneuploidy (fertilised egg missing that chromosome) can be taken as 50%.Similarly for biorientation, two single chromatids are present in thesecondary oocyte and these also segregate at random to either PB2 or thefertilised egg following meiosis. Thus, on this basis, the probabilityof both chromatids segregating to the polar body or oocyte is 25%(causing maternal monosomy and trisomy, respectively). If theprobability that one chromatid segregates to PB2 and one to thefertilised egg (euploid) is 50%, a PB1 chromosome exhibiting CH hasabout a 50% probability of resulting in aneuploidy.

If two or more chromosomes are identified as having CH the probabilitythat the fertilised egg is aneuploid for at least one of thosechromosomes can be calculated as a standard cumulative binomialprobability using the formula:

b(x;n,P)=nCx*Px*(1−P)n−x

wherex: The number of successes that result from the binomial experiment.n: The number of trials in the binomial experiment. P: The probabilityof success on an individual trial. 1-P: The probability of failure on anindividual trial.b(x; n, P): Binomial probability—the probability that an n-trialbinomial experiment results in exactly x successes, when the probabilityof success on an individual trial is P.nCr: The number of combinations of n things, taken r at a time.

Thus the probability of a fertilised egg with the following number of:chromosomes with CH having 1 aneuploidies is

1 0.52 0.753 0.8754 0.93757 0.96875

More generally, where one or more centromeres display a high proportionof heterozygous alleles (e.g. SNPs) indicating one or more CHchromosomes there is a higher probability that the corresponding egg (orfertilised egg, or embryo) is aneuploid, compared to a PB1 having alower degree of CH.

Where all centromeric regions display a low proportion of heterozygousalleles (e.g. SNPs) and it is inferred there are zero (0) CHchromosomes, there is a higher probability that the corresponding egg(or fertilised egg, or embryo) is euploid.

In practice, oocytes (which are subsequently fertilised normally) with 0CH chromosomes can be selectively transferred ahead of those with 1, 2or more CH-displaying chromosomes, with the aim of transferring thosewith an increased likelihood of having a normal number of maternalchromosomes increasing pregnancy rates and reducing miscarriage rates.

If desired, those skilled in the art can utilise morphokinetic or othermorphological analysis in conjunction with the assessment of risk madeusing total heterozygosity and\or CH, as well as crossover number and\orlocation analysis.

In summary, it can be seen that where there are a number of fertilisedeggs or embryos suitable for implantation, the present methods canassist in maximising the chance of selecting embryos which are euploid,and hence more likely to proceed to full term development withoutcongenital abnormalities.

Any of the methods or systems described herein may thus further comprisethe step of selecting an embryo or fertilised egg wherein PB1 exhibitsCH or high CH, or in which the PB1 CH otherwise indicates the presenceor likely presence of an aneuploidy in the embryo or fertilised egg.

Maternal Reference

As previously explained, the accuracy of detection of heterozygous SNPsin the regions flanking the centromeres is greatly improved by limitingthe SNP analysis to those SNPs which are heterozygous in the mother anddetermining which of these is heterozygous in PB1. As will be readilyappreciated, the mother's SNP genotype can be obtained from genomic DNAfrom a blood sample or discarded maternal cumulus cells which surroundthe egg when it is collected. Alternatively, given a number of PB1genotypes the maternal genotype can be reconstructed on the assumptionthat most pairs of maternal chromosomes will segregate normally in the1st meiotic division—for example wherever the genotype is AA in one ormore PB1 and BB in others, it is likely that the maternal genotype isAB.

Phasing of Maternal Heterozygous SNPs

This can be achieved using standard genetic analysis (see e.g. Kong,2008; Chowdhury, 2009) for example, the phasing of the two haplotypes onthe two homologous chromosomes can be ascertained by genotyping the DNAof the mother and comparing the results with one or more polar bodies.Both PB1 and PB2 should normally be homozygous for one or otherhaplotype and by examining several of these the haplotypes can bereconstructed with a high degree of accuracy.

Thus in one embodiment SNP haplotype (and hence phasing of heterozygousSNPs) is derived from analysis of multiple single haploid gametes orPB1s.

PB2 Reference

Although the method presented herein envisages analysing centromericSNPs (or genome-wide SNPs) in PB1 alone, and inferring the probabilityor relative risk of aneuploidy based on the abnormal condition ofheterozygosity, it will be appreciated that analysis of both PB1 and PB2for CH would provide confirmation of the euploid/aneuploid status ofchromosomes with CH in PB1.

Analysis of SNP Loci Across the Chromosome

In one embodiment, genome wide SNP arrays are used to genotype SNP locibeyond the region flanking the centromere, thereby extending intoregions where recombination is not supressed e.g. across the full lengthof each chromosome. Put another way, the primary analysis of SNP lociflanking each centromere, as explained in detail above, can be extendedalong each chromosome arm to give further information about ploidy.

In a normally segregating chromosome analysed in the PB1 followingcompletion of meiosis I, each chromosome is homozygous (at heterozygousmaternal loci) for one of the two maternal haplotypes in the regionflanking the centromere. Beyond the first crossover, however, one of thetwo sister chromatids has a segment from the other homologous chromosomeresulting in heterozygosity at all heterozygous maternal SNP loci. Thispattern of alternating homozygous and heterozygous bands is repeated asfurther crossovers occur down each arm. This results in a characteristicpattern for each chromosome according to the typical number and locationof each crossover.

Three abnormal patterns could be used to extend the CH analysis at thecentromeres:

The presence of two chromatids from one homologous chromosome with asister chromatid from the other homologue (caused by prematurepredivision) in PB1 will cause CH and the size of the homozygous bandsfurther down the chromosome will be reduced or non-existent compared tothe normal pattern because of a different pattern of crossovers (seeFIG. 3, reference 3.1 and 3.2 compared to FIG. 4, reference 4.1, 4.2,4.3).

The presence of both homologous chromosomes (all four sister chromatids)in PB1 will cause all maternal heterozygous SNP loci to be heterozygousalong the entire length of the chromosome (see FIG. 2, reference 2.5compared to 4.1, 4.2, 4.3).

The presence of only one sister chromatid in PB1 will result in all SNPloci being homozygous across the full length of the chromosomeindicating a risk of trisomy in the corresponding oocyte followingfertilisation (see FIG. 3, reference 3.3 and 3.4). It should be notedthat a homologous chromosome with no crossovers will give a similarpattern but this is also abnormal and liable to result in aneuploidy.

As noted above, assessing the number and location of crossovers has moredirect utility in establishing a tendency towards aneuploidy—for exampleif it is revealed that recombination frequency is low, or thatrecombination has occurred only near the centromere or telomere.

Interrogation of SNPs

Various methods for large scale single nucleotide polymorphism (SNP)analysis exist (see Syvanen, 2005, especially Table 1 therein). Theseinclude SNPstream (Bell, P. A. et al. SNPstream UHT: ultra-highthroughput SNP genotyping for pharmacogenomics and drug discovery.Biotechniques Suppl., 70-72, 74, 76-77 (2002)); Genorama, APEX (Kurg, A.et al. Arrayed primer extension: solid-phase four-colour DNAresequencing and mutation detection technology. Genet. Test. 4, 1-7(2000)); GeneChip 100K (Matsuzaki, H. et al. Genotyping over 100,000SNPs on a pair of oligonucleotide arrays. Nat. Methods 1, 109-111(2004)); Perlegen wafers (Hinds, D. A. et al. Whole-genome patterns ofcommon DNA variation in three human populations. Science 307, 1072-1079(2005)); Molecular Inversion Probes (Hardenbol, P. et al. Highlymultiplexed molecular inversion probe genotyping: Over 10,000 targetedSNPs genotyped in a single tube assay. Genome Res. 15, 269-275 (2005));GoldenGate Assay (Fan, J. B. et al. Highly parallel SNP genotyping. ColdSpring Harb. Symp. On Quant. Biol. LXVII, 69-78 (2003)). Other methodsinclude the Illumina “BeadArray” described below. It will also beunderstood that genome sequencing platforms (for example as availablefrom Illumina and others) may be used to establish heterozygositydirectly. So called “Next Generation Sequencing” platforms areparticularly preferred.

Preferred embodiments of methods for determining aneuploidy of polarbodies and oocytes disclosed herein comprise the use of microarraysystems. A microarray may include a population of different probemolecules that are attached to one or more substrates such that thedifferent probe molecules can be differentiated from each otheraccording to relative location. An array can include different probemolecules, or populations of the probe molecules, that are each locatedat a different addressable location on a substrate. Alternatively, amicroarray can include separate optical substrates, such as beads, eachbearing a different probe molecule, or population of the probemolecules, that can be identified according to the locations of theoptical substrates on a surface to which the substrates are attached oraccording to the locations of the substrates in a liquid. Exemplaryarrays in which separate substrates are located on a surface include,without limitation, a BeadChip Array available from Illumina®, Inc. (SanDiego, Calif.), for example the GoldenGate BeadChip arrays and InfiniumBeadChip arrays, or others including beads in wells such as thosedescribed in U.S. Pat. Nos. 6,266,459, 6,355,431, 6,770,441, 6,859,570,and 7,622,294; and PCT Publication No. WO 00/63437, each of which ishereby incorporated by reference. Other arrays having particles on asurface include those set forth in US 2005/0227252; WO 05/033681; and WO04/024328, each of which is hereby incorporated by reference.

Any of a variety of microarrays known in the art, including, forexample, those set forth herein, can be used. A typical microarraycontains sites, sometimes referred to as features, each having apopulation of probes. The population of probes at each site is typicallyhomogenous having a single species of probe, but in some embodiments thepopulations can each be heterogeneous. Sites or features of an array aretypically discrete, being separated with spaces between each other. Thesize of the probe sites and/or spacing between the sites can vary suchthat arrays can be high density, medium density or lower density. Highdensity arrays are characterized as having sites separated by less thanabout 15 μm. Medium density arrays have sites separated by about 15 to30 μm, while low density arrays have sites separated by greater than 30μm. An array useful in the invention can have sites that are separatedby less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm, or 0.5 μm.

Further examples of commercially available microarrays that can be usedinclude, for example, an Affymetrix® GeneChip® microarray or othermicroarray synthesized in accordance with techniques sometimes referredto as VLSIPS™ (Very Large Scale Immobilized Polymer Synthesis)technologies as described, for example, in U.S. Pat. Nos. 5,324,633;5,744,305; 5,451,683; 5,482,867; 5,491,074; 5,624,711; 5,795,716;5,831,070; 5,856,101; 5,858,659; 5,874,219; 5,968,740; 5,974,164;5,981,185; 5,981,956; 6,025,601; 6,033,860; 6,090,555; 6,136,269;6,022,963; 6,083,697; 6,291,183; 6,309,831; 6,416,949; 6,428,752 and6,482,591, each of which is hereby incorporated by reference. A spottedmicroarray can also be used in a method according to an embodiment ofthe invention. An exemplary spotted microarray is a CodeLink™ Arrayavailable from Amersham Biosciences. Another microarray that is usefulis one that is manufactured using inkjet printing methods such asSurePrint™ Technology available from Agilent Technologies.

The systems and methods set forth herein can be used to detect thepresence of a defined SNP that may be present in a PB1, embryo, oocyte,or derivative thereof.

A preferred embodiment employs the Affymetrix GeneChip™ 10K Microarraywhich is designed to analyse 10,000 SNPs distributed at an averagedistance of 0.2 Kb across each of 22 chromosomes (see Matsuzaki, H. etal. Parallel genotyping of over 10,000 SNPs using a one-primer assay ona high-density oligonucleotide array. Genome Res. 14, 414-425 (2004) Inthe case of oligonucleotide chips, the oligonucleotides that can bebonded to a chip according to the invention will be capable ofdistinguishing biallelic SNPs across the genome. Preferred are 25nucleotide-long oligonucleotides.

Thus in one embodiment the SNPs are interrogated on a “gene” or“oligonucleotide” chip or microarray. As is well known in the art theseare miniaturized vehicles, in most cases made of glass or silicon, onwhose surface oligonucleotides of known sequence are immobilized in anordered grid of high density.

Another preferred embodiment employs the Illumina Infinium™ HumanCytoSNP-12 Beadchip. This system enables genome-wide genotyping of about300,000 SNP markers. The system is based on the random assembly ofderivatized microscopic beads

-   -   approximately 3 μm in size placed into wells of a patterned        substrate, and permits specified combinations of SNPs to be        interrogated. The Infinium microarrays were utilized in Examples        of the disclosure, however any microarray platform appropriately        designed could also be utilized.

Amplification

As used herein, “amplification” refers to any process for multiplyingstrands of nucleic acid, such as genomic DNA, in vitro.

Amplification techniques include, but are not limited to, polymerasechain reaction (PCR) library based methods; and isothermal amplificationmethods, such as Multiple Displacement Amplification (MDA). Suchtechniques are well known to those skilled in the art—see for exampleAusubel et al., Short Protocols in Molecular Biology, (3rd ed.), Wiley &Sons; 2001, Sambrook and Russell, Molecular Cloning: A Laboratory Manual(3rd Edition); 1982, Maniatus et al., Molecular Cloning: A LaboratoryManual; DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.);1984; Spits, et al., 2006, Nature Protocols 1:1965-1970 “Whole genomemultiple displacement amplification from single cells”; U.S. Pat. No.6,124,120. Methods of whole genome amplification applicable to PGD arewell known to those in the art and have been reviewed by Ying-mingZheng, Ning Wang, Lei Li, and Fan in “Whole genome amplification inpreimplantation genetic diagnosis” J Zhejiang Univ Sci B. 2011 January;12(1): 1-11. doi: 10.1631/jzus.B1000196 PMCID: PMC3017410.

Systems

Preferably a system for use in the present invention would comprisemeans for SNP interrogation plus a programmed storage device or mediumfor causing a computer to analyse the resulting data. The SNPinterrogation data could be stored for later analysis or analysed ‘onthe fly’—as used herein the term “database” covers both types of datasource.

Preferred means for SNP interrogation would be an oligonucleotide chipwhich would interrogate at least the preferred chromosomes at theappropriate density in the vicinity of the centromere as discussedherein.

The methods disclosed herein may be implemented with the aid of acomputer. Typically this would include a central processing unit (CPU)connected by a system bus or other connecting means to a communicationinterface, system memory (RAM), non-volatile memory (ROM), and one ormore other storage devices such as a hard disk drive, a diskette drive,and a CD ROM drive.

The computer also includes a display device, such as a printer, CRTmonitor or an LCD display, and an input device, such as a keyboard,mouse, pen, touch-screen, or voice activation system. The input devicemay receive data directly from the means for SNP interrogation via aninterface (as for example with an Affymetrix or Illumina product basedsystem).

The computer stores and executes various programs such as an operatingsystem and application programs.

The computer-usable medium would cause the computer to analyse CH andassess the likelihood of aneuploidies of maternal origin in accordancewith the methods described herein. The medium may for example beselected from the group consisting of a hard disk, a floppy disk, RandomAccess Memory, Read Only Memory and Electrically Erasable ProgrammableRead Only Memory.

Thus the invention provides a computer-usable medium havingcomputer-readable program code or instructions stored thereon (i.e. aprogrammed storage device) for causing a computer to execute a methodassessing the risk of chromosomal aneuploidy of maternal meiotic originin a human egg following fertilisation, the method being any one ofthose discussed herein.

Preferably the method comprises:

-   (a) accessing a database comprising genotype data obtained from a    plurality of consecutive SNP loci present in a chromosome of the    PB1,-   (b) determining a centromere position,-   (c) determining the proportion of heterozygous SNPs in a region    flanking the centromere position by determining the number of SNPs    giving a heterozygosity call (n) compared to the total number of    SNPs (t) in the region;-   (d) determining that the corresponding fertilised egg or embryo is    at higher risk of aneuploidy when the proportion exceeds a reference    proportion.

Preferably the method comprises assessing the CH in a correspondingregion of the chromosomes from a maternal (e.g. somatic) cell. ThoseSNPs which are non-heterozygous in the maternal data are uninformative,and the method may be based purely on, or weighted towards, those lociwhich are heterozygous in the maternal data.

The method may additionally or alternatively utilise assessing SNP locipresent across a plurality of (preferably all) chromosomes of PB1 todetect unusually low levels of recombination, being indicative of atendency to aneuploidy.

The method may additionally or alternatively utilise assessing SNP locipresent across a plurality of (preferably all) chromosomes of PB1 todetect instances of single recombination events near the centromere ortelomere.

The invention also provides a computer programmed to execute a method asdescribed above.

It will be appreciated by those skilled in the art that the disclosedmethods, to the extent to which it entails analysing SNPs in PB1(optionally with PB2 also) to infer the nature of the maternalchromosomes in the fertilised egg or embryo, may be carried out inconjunction with other methodologies in which that data can also beutilised, or may be of interest, when determining aneuploidy or fordiagnosing of other diseases or conditions in a subject. Examplesinclude diagnosing disease by linkage, or diagnosing the presence orsusceptibility to a disease or cancer associated with particular SNPalleles or haplotypes—for example known single gene defects relevant todisease risk (see e.g. Table I of WO2011/138750 of the MRC et al).

Definitions

Allele—Each normal somatic cell has two copies of the genome on pairs ofhomologous chromosomes. A single copy of a gene or DNA marker, which maydiffer in sequence from the other copy, is referred to as an allele. Theterm “allele” is used consistent with its meaning in the art of biology.An allele is one or more alternative forms of a gene, genetic sequenceor single nucleotide (e.g. a single nucleotide polymorphism or SNP)found at a specific location, or locus, on a chromosome.

SNP—A single nucleotide polymorphism is a single base pair in the DNAsequence that varies between individuals. These occur frequentlythroughout the genome and are useful as markers. For example, biallelicSNPs can have either of two bases at a particular position which arereferred to generically as ‘A’ and ‘13’ herein.

Homologous chromosome—The human genome is duplicated in each cell on 23pairs of homologous chromosomes, one of each pair inherited from thefather and one from the mother.

Sister chromatid—DNA replication results in the duplication of eachchromosome into two sister chromatids which remain tightly boundtogether until they separate and segregate to opposite poles during celldivision.

Centromere—Specialised region of the chromosome which facilitates theattachment of spindle microtubules during cell division.

p arm—Term used for the shortest of the two arms of chromosomes withcentromeres placed more or less centrally (also ‘short arm’).

q arm—Term used for the longest of the two arms of chromosomes withcentromeres placed more or less centrally (also ‘long arm’).

Any sub-titles herein are included for convenience only, and are not tobe construed as limiting the disclosure in any way.

The invention will now be further described with reference to thefollowing non-limiting Figures and Examples. Other embodiments of theinvention will occur to those skilled in the art.

The disclosure of all references cited herein, inasmuch as it may beused by those skilled in the art to carry out the invention, is herebyspecifically incorporated herein by cross-reference.

FIGURES

FIG. 1—chromosome segregation in female meiosis I and II

(1) Female meiosis is initiated in the fetal ovary before birth duringthe early development of the female germ cells (primary oocytes), whichwill eventually form mature eggs or oocytes in the adult female. (2) Toreduce the number of chromosomes from the normal (euploid) 23 pairs ofhomologous chromosomes (one of each pair inherited from the father (lefthand side, including representative allele A) and one from the mother(right hand side, including representative allele B), or 46 in total, to23 single chromosomes, there is one round of DNA replication in whicheach chromosome is duplicated into two sister chromatids followed by twospecialised meiotic divisions, meiosis I and II. The two sisterchromatids have identical DNA sequences and therefore SNP genotypes atthis stage, including the regions flanking the centromere as representedhere in the boxes at the position of the centromeres, remain tightlybound together. (3) The two homologous chromosomes of each pair ‘pairup’ and a single bivalent chromosome forms in which all four sisterchromatids are tightly bound together. This allows a limited number ofbreaks in the DNA strands of adjacent non-sister chromatids to‘crossover’ and rejoin the other chromatid which results in the exchangeof a chromosome segment from that point to the end of the chromosomearm. (4) As the cell divides at the end of meiosis I, one homologouschromosome of each pair is pulled into the first polar body (left) andthe other into the secondary oocyte (right), which therefore now has 23chromosomes each with two sister chromatids. Note also that thehomologous chromosomes of paternal and maternal origin segregate atrandom according to Mendel's first law. In the diagram, a singlecrossover is illustrated between two non-sister chromatids. When the twohomologous chromosomes separate, therefore, there is one non-recombinantand one recombinant sister chromatid i.e. a chromatid which hasexchanged part of the chromosome with the other homologous chromosome.Note that because crossovers generally occur some distance away from thecentromere, at all maternal heterozygous positions flanking thecentromere, the two sister chromatids of both homologous chromosomes arehomozygous for one of the two maternal SNP alleles (copies). Beyond theposition of the crossover towards the ends of the chromosome some SNPcombinations on the two sister chromatids may be heterozygous. (5) Inmeiosis II, following fertilisation by a sperm cell containing thepaternal half set of chromosomes, the two sister chromatids of eachchromosome finally separate and segregate into the second polar body andfertilised oocyte or zygote. The zygote therefore inherits 23 singlematernal chromatids (or more simply at this stage ‘chromosomes’).

Errors in this normal pattern of chromosome duplication and segregationduring female meiosis can cause abnormal numbers of maternal chromosomes(aneuploidy) to be inherited in the zygote. Three basic mechanisms areknown to be associated with these errors and are illustrated in FIGS.2-4 and the euploid/aneuploid outcomes summarised in Table 1.

FIG. 2—female meiotic errors resulting in aneuploidy: (1) Classicalnon-disjunction of homologous chromosomes (Meiosis I) and sisterchromatids (Meiosis II)

Normally the two homologous chromosomes, which make up the singlebivalent chromosome, disjoin at the end of meiosis I, and move toopposite poles so that one homologous chromosome (with two sisterchromatids) ends up in the first polar body (PB1; left) and the other inthe secondary oocyte (right) (2.1). The classical textbook mechanismcausing aneuploidy in human oocytes is the non-disjunction of either thetwo homologous chromosomes in meiosis I (2.4 and 2.5), or the two sisterchromatids in meiosis II (2.2 and 2.3). Which homologous chromosomesegregates to the secondary oocyte is random so there are two possiblevariants of 2.1-2.3. This mechanism is now known only to occur in aminority of cases of aneuploidy.

FIG. 3—female meiotic errors resulting in aneuploidy: (2) Prematurepredivision of sister chromatids (Meiosis I)

Premature predivision of sister chromatids is now considered to be themain mechanism causing aneuploidy in the human oocyte. This is where oneof the two homologous chromosomes forming the single bivalent chromosomedisjoins normally and is pulled to one or the other pole. However, thetwo sister chromatids of the other homologous chromosome separateprematurely and one sister chromatid segregates to the first polar body(PB1) (left) and one to the secondary oocyte (right). At meiosis II, thetwo sister chromatids of the intact homologous chromosomes (if presentin the secondary oocyte) separate and segregate to the second polar body(PB2) and fertilised oocyte or zygote (as normal). The single sisterchromatid segregates at random to either PB2 or the zygote. The zygoteis therefore either euploid for that maternal chromosome (3.2 and 3.3)because the segregation of the single chromatid in meiosis II balancedthe error in meiosis I, or is aneuploid (3.1 and 3.4). As the intacthomologous chromosome randomly segregates either to PB1 or the secondaryoocyte and similarly that either sister chromatid of the prematurelypredividing homologous chromosomes can segregate to PB2 or zygote, thereare 4 possible variants of 3.1-3.4

FIG. 4—female meiotic errors resulting in aneuploidy: (3) Biorientationof both homologous chromosomes (Meiosis I)

The third mechanism causing aneuploidy is where the two sisterchromatids of both homologous chromosomes separate and segregate toopposite poles (biorient). In this case, two separate non-sisterchromatids segregate to the first polar body (PB1; left) and the othertwo to the secondary oocyte (right). At meiosis II followingfertilisation, both chromatids then segregate randomly to either poleand either one chromatid segregates to the second polar body (PB2)(right) and one to the fertilised oocyte or zygote (middle) (4.1) orboth segregate together to either PB2 or zygote causing aneuploidy (4.2and 4.3). Please note that because there are two possible combinationsof non-sister chromatids in meiosis I and because of random segregationof chromatids in meiosis II, there are 4 variants of 4.1 and two eachfor 4.2 and 4.3.

FIG. 5—flow chart

This Figure shows a flow chart illustrating a process for aneuploidyprediction by centromeric heterozygosity analysis based on disclosedmethods.

FIG. 6—centromeric heterozygosity detected in a first polar body (PB1)

For each autosome (1-22) and the X chromosome, the genotype ofconsecutive maternal heterozygous SNP loci flanking each centromere arerepresented by continuous columns in which the length is proportional tothe number of loci. In the figure yellow [lighter] and green [darker]indicate homozygous SNP loci for the two maternal haplotypes and redrepresents heterozygous SNP loci. The position of the centromere foreach chromosome is indicated by a light green band near the centre ofthe chromosome (or at the top of chromosomes 13-15 and 21-22).

Centromeric heterozygosity (CH) is present for chromosomes 4, 16 and 22with 72, 66 and 65% heterozygous AB loci, respectively, interspersedwith homozygous calls of either maternal haplotype caused by randomallele dropout. Similar analysis of the corresponding metaphase IIoocyte demonstrated that the CH for chromosomes 4 and 16 was caused bybiorientation and segregation of sister chromatids in both homologouschromosomes since CH was also observed. Whereas for chromosome 22,maternal heterozygous loci close to the centromere were homozygous inthe oocyte. This indicates that predivision of sister chromatids on onehomologous chromosome had resulted in three chromatids segregating toPB1 and only a single chromatid to the oocyte which could have resultedin monosomy following fertilisation. All of the other chromosomes arehomozygous in the region flanking the centromere for one of the twomaternal haplotypes (yellow or green) demonstrating independentinheritance of one of the mothers' chromosomes (single homologue withtwo sister chromatids) (Mendel's first law) with only an occasionalheterozygous miscall (these miscalls appear as one or more spaced-apart‘thin’ lines on various of the chromosomes i.e. 2, 6-12, 14, 17-21, x).

Three crossovers are detected away from (distal to) the centromeres inchromosomes 5, 6, and 17. It should be noted that only the regionflanking the centromere is shown so recombination is relativelyinfrequent. Beyond these crossovers a band of heterozygosity is presentsince one recombinant sister chromatid now has a segment from the otherhomologous chromosome. Again analysis of the corresponding metaphase IIoocyte confirmed the presence of the other homologue (with two sisterchromatids) with homozygous SNP loci for the opposite maternal haplotypeand the same heterozygous banding pattern.

The apparent change from homozygosity from one haplotype to the other inchromosome 7 (green to yellow) is an artefact and results from acrossover in the reference genotype used to phase the A and B maternalalleles. This apparent complete step-change in the maternal haplotypecan be readily detected and distinguished from the quite differentpattern of calls resulting from true CH or crossover distal to thecentromere, and can thus be ignored in the analysis.

FIG. 7—Mapping recombination in all three products of meiosis in adulthuman oocytes

(a) Crossing over and bivalent formation between homologous chromosomesduring foetal development. Upon oocyte maturation, homologouschromosomes (defined by pericentromeric genetic markers) segregate andPB1 is formed. These mature MII oocytes arrest again, before beingactivated by sperm (or Ca2+ ionophore) and completing the second meioticdivision. To the right, a brightfield image of an activated oocyte withPB2 is shown.

(b) Schematic of MDA amplification of DNA from PB1, PB2 and oocyte inthree separate reactions followed by hybridization to the IIlumina 300KSNP array. The readout from the SNP array is shown at the bottom. Thehaplotype breakpoint can be seen in the oocyte and a reciprocalheterozygous block is present in the PB1. Note that the SNPs in thisregion of PB1 cannot be phased.

(c) Schematic of chromosome showing crossovers, which can be ascertainedby heterozygosity analysis of PB1 (arrowheads, left hand side) and thoserequiring PB2 or oocyte analysis (arrow, right hand side).

FIG. 8—Crossover distribution and crossover tract lengths in humanfemale meiosis.

(a) Recombination events estimated from the oocyte-PB trios (Trio),oocyte only, PB1 only and PB2 only in one to four oocytes from fivedifferent donors.

(b-e) Spearman correlation (p) between crossover levels based on Trioand PB1, Trio and oocyte, trio and PB2 and oocyte and PB2. Note thatWilcox tests gave significant values, but not for (e).

FIG. 9—Histogram summarising results of Table 2 in Example 3

The histogram clearly demonstrates the relationship between the PB1heterozygosity pattern and embryo karyotype.

EXAMPLES Example 1—Meiotic Errors that can Lead to Aneuploidy in aFertilized Egg

As illustrated in FIG. 1, normally in meiosis I the two homologues ofeach chromosome, now consisting of pairs of sister chromatids, pair upand join together, homologous non-sister chromatids undergo one or morerecombinations or crossovers.

Subsequently the homologous chromosomes of the condensed bivalentchromosome are ‘co-oriented’ to the same spindle poles, so that thehomologous chromosomes separate away from each other into PB1 and thesecondary oocyte. In meiosis II, the two sister chromatids separate intoPB2 and fertilised oocyte (zygote or egg) following fertilisation.

It can thus be seen that in the in the normal (euploid) oocyte allcentromeric regions of each chromosome are homozygous in PB1, since thispart of the sister chromatids will generally not be significantlyaffected by recombination or crossovers.

The primary oocyte in the fetal ovary has 23 pairs of homologouschromosomes (46 in total), one inherited from the father and one fromthe mother, as in somatic cells.

-   -   1001—Representative maternal heterozygous (AB) SNP close to        centromere.    -   1002—Pair of homologous chromosomes (blue and red)    -   1003—Primary oocyte

Following DNA replication early in meiosis I both homologous chromosomesconsist of pairs of sister chromatids closely bound together, withidentical duplicated SNPs.

-   -   1004—Short or ‘p’ arm    -   1005—Position of centromere    -   1006—Long or ‘q’ arm    -   1007—Pair of sister chromatids

Homologous chromosomes pair and all four sister chromatids are closelybound together into 23 thread-like bivalent chromosomes. The chromatidsbreak and non-sister chromatids join up and exchange parts of chromosomearms at crossovers.

-   -   1008—Single bivalent chromosome (shown diagrammatically in 2D    -   1009—Single crossover on the long arm between two non-sister        chromatids

Prior to ovulation in the adult, the maturing oocyte completes meiosisI. The two homologous chromosomes within each bivalent segregate intothe first polar body (PB1) and secondary oocyte which arrests inmetaphase of meiosis II.

-   -   1010—First polar body (PB1)    -   1011—Secondary oocyte    -   1012—Exchanged chromosome segments from the position of the        crossover to the end of the chromosome arm

At fertilization, meiosis II is completed and the two sister chromatidsin the secondary oocyte segregate into the second polar body (PB2) andfertilized oocyte or zygote reducing maternal chromosome number from 46to 23 (paternal chromosomes not shown).

-   -   1013—Single chromatids (or simply chromosomes)    -   1014—Second polar body (PB2)    -   1015—Fertilised oocyte or zygote

As illustrated in FIG. 2, a classical textbook mechanism causing errorsin meiosis is ‘non-disjunction’ of homologous chromosomes in meiosis Iand sister chromatids in meiosis II.

Where this kind of error has occurred, analysis of CH or loss ofcentromeric DNA in PB1, would detect these errors in meiosis I. Thistype of error is now known to be rare.

As illustrated in FIG. 3, another source of errors is ‘prematurepredivision of sister chromatids’ in meiosis I. Copy number analysis byarray CGH has confirmed this is the predominant mechanism causinganeuploidy in advanced maternal age (Handyside et al., 2012). Gain of asister chromatid would be detected by CH in PB1 but loss of a sisterchromatid would not be detected as the centromere would appear to behomozygous.

All of the above errors in meiosis I can be detected by quantitationusing prior art methods such as 24Sure™ (BlueGnome, Cambridge UK)following manufacturer's protocol.

As illustrated in FIG. 4, it has also been determined that errors mayarise in meiosis I whereby both homologous chromosomes ‘bi-orient’sister chromatids to opposite spindle poles and separate at meiosis I,in a ‘mitosis-like’ division. The two unpaired sister chromatids thenare likely to segregate randomly to either pole at meiosis II and in 50%of cases, both sister chromatids will either segregate to PB2 or theoocyte resulting in aneuploidy. Furthermore it has been shown that thisincreased risk of aneuploidy in the resulting fertilised egg followingmeiosis II, arising from the original separation of sister chromatids inmeiosis I, can be detected by CH in PB1.

In particular normal euploid oocytes would be expected to have allcentromeric heterozygous loci homozygous in PB1, whereas one or morecentromeres heterozygous or missing in PB1 would imply an at-riskoocyte.

In will be understood that combining detection of CH in PB1 withquantitation would be fully informative for meiosis I errors (see FIGS.1(3)) while also being able to assess risk of meiosis II errors evenwhere there is an apparently normal (euploid) PB1.

Example 2—Assessment of CH in PB1 from Number of Different Oocytes

In this example the status of defined SNPs were assessed in PBs (“PB1genotype”) and corresponding secondary oocytes (“Egg genotype”).

Methods

PBs and oocytes were lysed and the whole genome amplified (WGA) bymultiple displacement amplification according to manufacturer'sinstructions (Repli-g, Qiagen). WGA products were then genotyped on aSNP genotyping bead array again according to the manufacturer's protocol(Infinium Human CytoSNP-12, IIlumina). The genotype data was exported asa text file and imported into Microsoft Excel and a macro was used toidentify SNPs flanking the centromeres of each chromosome and displaythe results. The macro also calculated the percentage of heterozygousSNPs.

The maternal genotype was ascertained using the same bead array butgenomic DNA isolated from a blood sample by standard methods was used.This data was also imported into Excel and used to identify all of theheterozygous SNPs in the regions of the centromeres so that thesubsequent analysis of the PB1s could be limited to only these loci.

Using this method 200 SNPs flanking each of the centromeres of the 18non-acrocentric chromosomes and on the q arm of the 5 acrocentricchromosomes for a total of 8200 defined SNPs (18×400+5×200) wereassessed in PBs and secondary oocytes.

Theoretical Considerations

Theoretically, the abnormal presence of two non-sister chromatids, oneeach from the two homologous chromosomes, should result in allheterozygous maternal SNP loci flanking the centromere to the positionof the first crossover also being heterozygous in PB1. Analysing onlymaternal heterozygous loci (as shown in FIG. 6) would therefore resultin a continuous series of heterozygous SNPs to the point of thecrossover, when all of the SNPs would be homozygous.

These homozygous SNPs beyond the cross over, will be consistent with oneor the other of the maternal haplotypes (if the maternal SNPs arephased).

In practice, when genotyping single cells following whole genomeamplification, failure to amplify one of the two SNP copies at eachheterozygous locus (allele dropout or ADO) is relatively frequent (up toapproximately 50%). Therefore the expected pattern in PB1 for theabnormal situation is a chromosome with a high incidence of heterozygousloci, including around the centromere (CH), but interspersed withhomozygous SNPs of either maternal haplotype at random.

Beyond the crossover there may still be the occasional genotyping error(‘miscalls’ or ‘drop-ins’) resulting in a homozygous SNP to be called asheterozygous (approximately 5-10%).

Results

The results are shown in FIG. 6 for one egg (termed ‘Egg9’). Data showedthere were relatively low levels (<=5%) of AB ‘miscalls’ or ‘drop-ins’for most chromosomes (i.e. a low incidence of where the mother wasdetermined not to be heterozygous, but the PB1 associated with Egg9 wascalled as heterozygous).

A high level of centromeric heterozygosity (CH) for chromosomes 4, 16and 22 was found when evaluating PB1 associated with Egg9

The three affected chromosomes (4, 16 and 22) demonstrate thetheoretical pattern explained above, with a high incidence ofheterozygous loci close to the centromere interspersed with homozygousSNPs of either maternal haplotype at random due to ADO.

As can be readily seen in FIG. 6, the presence of this ADO did notprevent the accurate detection of CH.

Similar analysis of the corresponding metaphase II oocyte (not shown)demonstrated that two of these (4 and 16) were caused by biorientationof both homologues so that the centromeres in the oocyte were alsoheterozygous. For chromosome 22 the CH was caused by prematurepredivision of sister chromatids with an extra chromatid being inheritedin PB1 and the oocyte chromosome was homozygous in the region of thecentromere. The former would therefore have normal copy number and theaneuploidy risk would not be detected by array CGH.

Additionally, chromosome 17 had elevated heterozygous levels, but thiswas found to be due to a crossover close to the centromere on the q arm,which resulted in distal heterozygosity (i.e. away from the centromere)which could be readily distinguished from the affected chromosomes.

Prognostic Use

Due to presence of a high level of CH in the PB1 associated with Egg9,Egg9 can be classified as higher risk than a corresponding Egg in whichCH was lower in its associated PB1 or absent.

In conclusion, chromosomes 4 and 16 demonstrated high CH in bothproducts of meiosis I (PB1 and secondary oocyte) of Egg9. Bothhomologues of these chromosomes have divided in a ‘mitosis-like’ wayresulting in one chromatid for each homologue in both PB1 and meiosis IIoocyte. It is notable that as the copy number of these chromosomes isnormal in PB1 (2n chromatids) this is not detected by array CGH.However, as there is no DNA replication in meiosis II, the two singlechromatids will theoretically segregate at random resulting in a 50%risk of aneuploidy in the fertilised oocyte (FIG. 4).

With chromosome 22, there is a high level of CH in PB1 but not thesecondary oocyte. This indicates chromatid gain in PB1 and a 50% risk ofloss in the fertilised oocyte (cf. FIG. 1(3)). In absence of data fromthe secondary oocyte, this could be confirmed by quantitative analysisof PB1.

The cumulative binomial probability that at least one of thesechromosomes will result in aneuploidy is therefore 0.875.

Thus it can be seen that assessing the presence or degree of centromericheterozygosity in chromosomes of the first polar body of the egg can beused to assess the risk of chromosomal aneuploidy of maternal meioticorigin.

Example 3—Comparison of Array CGH for Quantitative Detection ofAneuploidy in PB1 and PB2 with SNP Genotyping, Maternal Haplotyping andTotal Heterozygosity and/or CH Analysis in PB1 Alone, or PB1 and PB2

Ten mature MII arrested oocytes were collected from a patient havinganeuploidy testing by array CGH of PB1 and PB2. PB1 was biopsied fromeach oocyte prior to intracytoplasmic sperm microinjection and,following fertilisation and resumption of meiosis, PB2 was alsobiopsied. Both polar bodies were lysed, DNA amplified by WGA andaliquots of the products used for array CGH. The array CGH resultsindicated that all of the embryos had one or more copy numberabnormalities in PB1 and/or PB2 except one, presumed euploid embryo(Embryo #1), which was therefore selected for transfer (Table 2). Withthe patients consent, the remaining 9 presumed aneuploid embryos werelysed and the DNA amplified by WGA. Genomic DNA from both parents, WGAproducts from all polar bodies and the corresponding embryos were thenSNP genotyped. A combination of (1) maternal haplotyping using a PB2 asa reference to phase the heterozygous maternal SNPs, (2) heterozygosityanalysis, and (3) haplotyping of both parental chromosomes in the embryo(Handyside et al, 2010) was then used to analyse the pattern ofcrossovers genome wide in PB1, PB2 and the corresponding embryos.

SNP analysis of the PBs and the corresponding embryos were 100%concordant and confirmed that 7/9 embryos predicted to be aneuploid byarray CGH of PB1 and PB2, were aneuploid. However, the remaining twoembryos (Embryos #2 and 12; Table 2) with three and multiple copy numberabnormalities, respectively, identified by array CGH were euploid (Table2; see also FIG. 9). Furthermore, no abnormal SNP patterns, includingtotal and/or centromeric heterozygosity, were detected for any of thechromosomes in these two euploid embryos. In contrast, 19 examples ofabnormal SNP patterns occurred in the 7 aneuploid embryos (boldindicates an aneuploid outcome in the corresponding embryo):

-   -   1× MI NDJ (see FIGS. 2.4, 2.5)    -   3× PD (1× balanced (see FIG. 3.2), 2× unbalanced (FIG. 3.4))    -   15× BO (11× balanced, 4× unbalanced; see FIGS. 4.1 vs. 4.2, 4.3)

The 11× balanced BO were all confirmed as having opposite centromerichaplotypes in PB2 and embryo (FIG. 4.1).

Therefore, whereas array CGH analysis of PB1 alone would have predictedthe possibility of 3/7 aneuploidies, analysis of the distribution ofheterozygous SNPs in PB1 alone included abnormalities for 7/7 of theaneuploid chromosomes plus 12 other abnormalities associated withincreased risk of aneuploidy. This demonstrates the utility of using PB1heterozygosity assessment for diagnostic or prognostic screening.

More specifically 7/7 aneuploidies were predicted based on analysis ofhaplotypes in:

9× cen het (embryo 7, 8, 10, 11, 13, 14, 16)->1× MI trisomy 1× monosomy7× hetero (embryo 8, 10, 13, 14)->2× monosomy 2× homo (embryo 7, 10)->2×MI trisomy1× loss (embryo 11)->1× MI trisomy

TABLE 2 Example data comparing array CGH analysis of chromosome copynumber in PB1 and PB2 with SNP genotyping and maternal haplotyping, andparental haplotyping (karyomapping) of the corresponding embryos. ArrayCGH Mat haplotyping Type of Karyomapping Embryo # PB1 PB2 PB1 PB2abnormality of embryo 1 Euploid Euploid Euploid Euploid None Transferred2 +6, +10, +18 Euploid Euploid Euploid None Euploid 7 −22 Euploid 16-cenhet Euploid 16-BO bal +22 Ml 21-cen het 21-BO bal 22-homo 22-PD unbal 8+15 −9, −15 9-cen het −9, −15 9-BO unbal  +9 Ml 15-hetero 15-PD bal 10−15, −19, −22 Euploid 15-homo Euploid 15-PD unbal +15 Ml 16-hetero 16-BObal 19-hetero 19-BO bal 21-cen het 21-BO bal 22-cen het 22-BO bal 11 −22+22 4-cen het 22-cen het 4-BO bal +22 Ml −22 22-MI NDJ 12 EuploidMultiple copy Euploid Euploid None Euploid number abnormalities 13Euploid del 4, +22 16-cen het del 4 16-BO bal −22 Mil 21-hetero22-hetero 21-BO bal 22-hetero 22-BO unbal 14 Euploid −10, −13, +19, −2017-cen het 19-hetero 17-BO bal −19 Mil 19-hetero 19-BO unbal 22-hetero22-BO bal 16 Euploid +21 21-cen het 21-cen het 21-BO unbal −21 Mil NDJ =Non-disjunction (see FIG. 2); PD = Premature Predivision (see FIG. 3);BO = Biorientation (see FIG. 4); Euploid (a normal number of maternalchromosomes); Del = partial deletion; Bal = balanced; Unbal =unbalanced; NR = No results|

Example 4—Meiotic Recombination Analysis

It is known in the art that a low incidence and/or abnormal pattern ofrecombination close to the centromere or telomere is associated withabnormalities in chromosome segregation leading to aneuploidy (seeHassold, 2001). For example in the human population, the US NationalDown Syndrome project have reported that the rate of chromosomes 21lacking recombination events is enriched in patients with Trisomy 21compared to healthy individuals.

A similar analysis to that described above for Example 3, was carriedout with 13 oocytes from 5 patients. However, in this example, followingbiopsy of PB1, resumption of MII and extrusion of PB2 was initiated byartificially activating the oocytes (FIG. 7a ). Both polar bodies andthe activated oocyte were then lysed, DNA amplified by WGA and SNPgenotyped together with genomic DNA from the patient (FIG. 7b ). Usingthe genotype of one of the PB2s or eggs, the two maternal haploptyeswere then ascertained and analysed along with the pattern ofheterozygosity in PB1, PB2 and the corresponding activated oocytes. Thisenabled genome-wide maps of meiotic recombination by analysis of thematernal haplotypes present.

Crossovers (recombination) between the homologous chromosomes (i.e.between two non-sister chromatids) results in a switch in haplotypedistal to the haplotype breakpoint in both PB1 and either the PB2 oregg, but not both (FIG. 7c , arrowheads). In PB1, which normally retainsboth sister chromatids for one homologue, this results in heterozygositydistal to the first breakpoint (blue) followed by alternating bands ofhomozygosity and heterozygosity at successive crossovers towards thetelomere of each chromosome arm. Whereas in PB2 or egg, which normallyhas only a single chromatid from the other homologue, this results in aswitch from one haplotype to the other (yellow to green or vice versa)and the entire chromosome is homozygous. Thus, the number and positionof these crossovers can be ascertained by analysing the number andposition of regions of heterozygosity in PB1 alone. In addition,however, crossovers can also occur between sister chromatids in regionswhere a proximal crossover resulted in sister chromatids with oppositehaplotypes. These can only be detected by examining the pattern ofhaplotype switching in PB2 and/or oocyte (FIG. 7c , arrow) since theyonly involve one of the two homologous chromosomes. In the correspondingheterozygous regions of the other homologous chromosome in PB1, thesecrossovers cannot be detected at all by genotype analysis, since bothsister chromatids (with opposite haplotypes) switch haplotypes and thechromosome remains heterozygous. Thus the closest estimate of theposition and total number of crossovers for each chromosome is the sumof those which can be detected by PB1 analysis alone and those occurringonly in PB2 and oocyte.

Analysis of the pattern of heterozygosity and maternal haplotypes in the13 activated oocytes revealed 920 crossovers in the 23 chromosomesanalysed (22 autosomes and the X chromosome). There was a wide variationin the overall number of crossovers detected in PB1, PB2 and oocyte andtotal number (trio) in individual oocytes (FIG. 8a ).

However, the number of crossovers in PB1 was highly correlated with thetotal number combining analysis of PB1, PB2 and oocyte (‘Trio’) (FIG. 8b).

Thus the number of crossovers in PB1, which is determinable using themethods described herein can be used as a tool or additional tool forassessing risk of aneuploidy in the oocyte. That in turn can be used inoocyte or embryo selection and/or as a prognostic indicator oflikelihood of pregnancy.

Example 5—High Resolution SNP Mapping of PB1 can Reveal De NovoStructural Chromosome Abnormalities

In humans, chromosomal abnormalities either in structure or numberscontribute towards disorders, infertility, and pregnancy loss (Nagaoka,2012).

The analysis by the inventors of all products of meiosis described inExamples 3 and 4 above, allowed them to discern de novo alterations tochromosomes that could be specifically assigned to the human femalegermline.

For example in the data described in Example 4, three major structuraldefects (0.5% of chromosomes) from three different oocytes, wereobserved, all of which included imbalance in the oocyte itself.

One consisted of the gain of small piece of the q-arm of chromosome 22(1.5 Mb) concomitant with the loss of the majority of the q-arm in theoocyte. Both the gain and loss was reflected in the PB1. The losseffectively leads to an aneuploid oocyte and would be predicted to causeembryo loss. In human, even a relatively small deletion of 22q13.3 isassociated with Phelan-McDermid syndrome (Phelan, 1993).

The second structural change was the addition of 9.2 Mb of the p-arm ofchromosome 8 in the oocyte, with concomitant loss in the PB1. This couldeither be an inversion—duplication or a non-chromosomally associatedfragment of chromosome 8. Gain of 8p is associated withmyeloproliferative syndrome (Macdonald, 1995).

Finally, nearly 2/3 of the q-arm of chromosome 15 was present twice in athird oocyte, with a concomitant loss in the PB1.

These observations support the conclusion that the structural defectsarose during meiosis in female germline, and that PB1 can be used toobserve or infer the possible presence of such defects.

REFERENCE LIST

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1. (canceled)
 2. A system for assessing centromeric heterozygosity ofmaternal meiotic origin in a human egg, the human egg comprising a firstpolar body (PB1), the first polar body comprising one or more PB1chromosomes, the system comprising: (i) means for performing a nucleicacid detection assay to interrogate at least 25 biallelic SNPs flankingthe centromeres of said one or more PB1 chromosomes, said SNPs locatedwithin 5 to 10 Mb of the centromere; and (ii) a programmed storagedevice or medium comprising instructions assessing the presence ordegree of centromeric heterozygosity (CH) for said one or more PB1chromosomes, based on the results of the nucleic acid detection assay.3. The system of claim 2, wherein the means for SNP interrogation is amicroarray.
 4. The system of claim 2, wherein the means for SNPinterrogation is a sequencing platform.
 5. The system of claim 2,further comprising instructions for determining the risk that the humanegg will give rise to an aneuploid fertilised egg or embryo followingmeiosis II, wherein the presence or a higher level of CH in the firstpolar body indicates a higher risk of said chromosomal aneuploidy in thecorresponding fertilised egg or embryo developed therefrom compared towhere an absence or lower level of CH is present in the first polarbody.
 6. The system of claim 2, further comprising instructions forassessing the total number of crossovers in the PB1 chromosomes, basedon the heterozygosity of some or all of the chromosomes of the firstpolar body of the egg, and determining the risk that the human egg willgive rise to an aneuploid fertilised egg or embryo, wherein a belowaverage total number of crossovers determined in the PB1 chromosomesindicates a higher risk of aneuploidy, compared to where an averagenumber of crossovers is determined in the PB1 chromosomes.
 7. The systemof claim 2 further comprising instructions for assessing the position ofcrossovers in said one or more of the PB1 chromosomes, based on theheterozygosity of one or more of the chromosomes of the first polar bodyof the egg, and determining the risk that the human egg will give riseto an aneuploid fertilised egg or embryo, wherein a PB1 chromosomehaving only a single crossover proximal to the telomere or centromereindicates a higher risk of aneuploidy, compared to a PB1 chromosomehaving a plurality of medially distributed crossovers.
 8. The system ofclaim 2 further comprising instructions for assessing the presence ofstructural defects in said one or more of the PB1 chromosomes, based onthe heterozygosity of one or more of the chromosomes of the first polarbody of the egg, and determining the risk that the human egg will giverise to an aneuploid fertilised egg or embryo, wherein a PB1 chromosomeshowing a structural defect indicates a higher risk of aneuploidy. 9.The system of claim 8 wherein the structural defects are chromosomal orsub-chromosomal defects selected from: gains, losses, and duplications.10. The system of claim 2 further comprising instructions fordistinguishing (i) a polar body which contains a chromosome consistingof sister chromatids replicated from one of the homologous chromosomesfrom the mother, from (ii) a polar body wherein the chromosomes comprisetwo or more non-sister chromatids being collectively derived from bothof the homologous maternal chromosomes, and determining the risk thatthe human egg will give rise to an aneuploid fertilised egg or embryo,wherein (ii) indicates a higher risk of chromosomal aneuploidy ofmaternal meiotic origin in the corresponding egg.
 11. The system ofclaim 2 further comprising instructions for assessing a plurality ofdifferent first polar bodies such as to grade the corresponding eggs, orfertilised eggs or embryos developed therefrom, according to their riskof possible chromosomal aneuploidy of maternal meiotic origin.
 12. Thesystem of claim 2 wherein the polar body is from a human female who haspreviously been diagnosed as having fertility problems or having orcarrying an inheritable disease.
 13. The system of claim 2 wherein thepolar body is from a human female who is undergoing IVF treatment. 14.The system of claim 20 further comprising instructions for determiningthe risk of possible chromosomal aneuploidy of maternal meiotic origin,and based on said risk of possible chromosomal aneuploidy, determining alikelihood of pregnancy in said human female.
 15. The system of claim 2wherein at least 5, 10, 15 or 20 chromosomes are assessed, or optionallyall 23 chromosomes are assessed per polar body.
 16. The system of claim2 wherein centromeric heterozygosity is assessed for 2 or more of thehuman chromosomes selected from the group consisting of: X, 22, 21, 18,16 and
 13. 17. The system of claim 2 further comprising a means forwhole genome amplification of said first polar body prior to saidnucleic acid detection assay.
 18. The system of claim 2 wherein saidinstructions for assessing the presence or degree of centromericheterozygosity (CH) comprise: (i) phasing the SNPs of the maternalchromosomes; and (ii) based on the SNP phasing from step (i),determining the presence of centromeric heterozygosity (CH) from anybiallelic SNPs which are heterozygous maternal loci, but wherein saidnucleic acid detection assay gives a homozygous call due to randomallele dropout.
 19. The system of claim 2 wherein equal to or at least30, 40, 50, 75, 100, 200, 300, 400, 500 or more SNPs are interrogatedflanking the centromere, wherein SNPs on both of the p and q arms ofnon-acrocentric chromosome are assessed.
 20. The system of claim 2wherein said instructions for assessing the presence or degree ofcentromeric heterozygosity (CH) comprises quantifying a proportion ofheterozygous SNPs.
 21. The system of claim 20, further comprisinginstructions for comparing the proportion of heterozygous SNPs in thefirst polar body to a proportion of heterozygous SNPs in a maternal cellgenotype, wherein the maternal cell genotype comprises at least 10, 15,20, 25, 50, 100 or more heterozygous SNPs.